The long-term goal of this project is to understand the molecular mechanism by which proteins are transported across or are inserted into the mammalian endoplasmic reticulum (ER) membrane. We have established reconstituted proteoliposome and soluble systems with purified membrane protein components which reproduce the overall translocation process and partial reactions. The minimum translocation apparatus of the ER membrane comprises only the heterotrimeric Sec61p complex, the TRAM protein, and the SRP receptor. We now propose to perform a thorough analysis of the mechanism of translocation. Specifically, we will use questions: 1. How is cotranslational translocation initiated? 2. How do ribosomes interact with the Sec61p channel? 3. How are membrane proteins inserted into the lipid bilayer? 4. What is the function of the mammalian homologs of Sec62p and Sec63p? These studies will contribute significantly to our understanding of the first and decisive step in the biosynthesis of a large of class proteins, proteins of the plasma membrane, of lysosomes, and of all organelles of the secretory pathway.